Monogenic Mutations Research Articles

Polygenic score: An anchor holding the whole life course.

Polygenic score: An anchor holding the whole life course.

Association of polygenic risk score with response to deep brain stimulation in Parkinson’s disease

BackgroundDeep brain stimulation (DBS) is a well-established treatment option for select patients with Parkinson’s Disease (PD). However, response to DBS varies, therefore, the ability to predict who will have better outcomes can aid patient selection. Some PD-related monogenic mutations have been reported among factors that influence response to DBS. However, monogenic disease accounts for only a minority of patients with PD. The polygenic risk score (PRS) is an indication of cumulative genetic risk for disease. The PRS in PD has also been correlated with age of onset and symptom progression, but it is unknown whether correlations exist between PRS and DBS response. Here, we performed a pilot study to look for any such correlation.MethodsWe performed a retrospective analysis of 33 PD patients from the NIH PD Clinic and 13 patients from the Parkinson’s Progression Markers Initiative database who had genetic testing and underwent bilateral subthalamic nucleus DBS surgery and clinical follow-up. A PD-specific PRS was calculated for all 46 patients based on the 90 susceptibility variants identified in the latest PD genome-wide association study. We tested associations between PRS and pre- and post-surgery motor and cognitive measures using multiple regression analysis for up to two years after surgery.ResultsChanges in scores on the Beck Depression Inventory (BDI) were not correlated with PRS when derived from all susceptibility variants, however, when removing pathogenic and high-risk carriers from the calculation, higher PRS was significantly associated with greater reduction in BDI score at 3 months and with similar trend 24 months after DBS. PRS was not a significant predictor of Unified Parkinson’s Disease Rating Scale, Dementia Rating Scale, or phenomic and semantic fluency outcomes at 3- and 24-months after DBS surgery.ConclusionsThis exploratory study suggests that PRS may predict degree of improvement in depressive symptoms after DBS, though was not predictive of motor and other cognitive outcomes after DBS. Additionally, PRS may be most relevant in predicting DBS outcomes in patients lacking pathogenic or high-risk PD variants. However, this was a small preliminary study and response to DBS treatment is multifactorial, therefore, more standardized high-powered studies are needed.

An Update of Clinical, Epidemiological, and Psychosocial Features in Gamma-Sarcoglycanopathy

Limb-girdle muscular dystrophies (LGMDs) represent a group of muscle diseases due to monogenic mutations encoding muscle proteins that are defective for heterozygous and homozygous mutations prevalent in certain regions. Advances in knowledge of their pathophysiology have shed light on these rare diseases, which were, until recently, difficult to diagnose. This paper has described the process of diagnosis in autosomal recessive limb-girdle dystrophy that in Tunisia are due to the c.521del mutation in gamma-sarcoglycanopathy and to ethnically specific mutations in other countries such as Italy. The epidemiology, pathophysiology clinical features, and the main socioeconomic needs as well as research progress are discussed. We discuss an Italian case for its psychosocial impact and socioeconomic consideration and compare this case with Tunisian patients.

MP24-12 ASSOCIATION OF RARE MONOGENIC VARIANTS WITH DIAGNOSIS OF BPH USING WHOLE EXOME SEQUENCING IN THE UK BIOBANK

You have accessJournal of UrologyCME1 Apr 2023MP24-12 ASSOCIATION OF RARE MONOGENIC VARIANTS WITH DIAGNOSIS OF BPH USING WHOLE EXOME SEQUENCING IN THE UK BIOBANK Clark Judge, David Nusbaum, Jun Wei, Zhuqing Shi, Andrew Rifkin, S. Lilly Zheng, Brian Helfand, Alexander Glaser, and Jianfeng Xu Clark JudgeClark Judge More articles by this author , David NusbaumDavid Nusbaum More articles by this author , Jun WeiJun Wei More articles by this author , Zhuqing ShiZhuqing Shi More articles by this author , Andrew RifkinAndrew Rifkin More articles by this author , S. Lilly ZhengS. Lilly Zheng More articles by this author , Brian HelfandBrian Helfand More articles by this author , Alexander GlaserAlexander Glaser More articles by this author , and Jianfeng XuJianfeng Xu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003249.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Benign prostatic hyperplasia (BPH) is a common condition that leads to bothersome lower urinary tract symptoms. Polygenic heritability of BPH has been demonstrated using genetic risk scores derived from common single nucleotide polymorphisms, but rare monogenic gene mutations associated with BPH have not been well described. The objective of this study was to determine the impact of rare monogenic variants on the development of BPH. METHODS: A total of 83,631 men from the UK Biobank (UKB) with whole exome sequencing data were included and categorized based on ICD10 diagnosis of BPH (N40). Of men with BPH, 9,697 were further characterized as undergoing transurethral resection of prostate (TURP). The robust SKAT-O, a novel gene-based analysis of pathogenic/likely pathogenic mutations that properly controls for type I error rates due to unbalanced case-control ratio, was used for association tests adjusting for age at recruitment and genetic background. RESULTS: Nineteen candidate genes were identified as associated with BPH in 9,697 cases compared to 73,934 controls at p<0.001 using SKAT-O (Table). However, none of these associations was significant after adjusting for multiple testing (FDR<0.05). For most of these genes, higher carrier rates in patients undergoing TURP was also found. CONCLUSIONS: In this large study of nearly 84,000 men, several candidate genes were identified. Confirmation in independent study populations is required. Identification of monogenic gene mutations associated with BPH may advance our understanding of disease etiology and identify potential therapeutic targets. Source of Funding: NA © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e322 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Clark Judge More articles by this author David Nusbaum More articles by this author Jun Wei More articles by this author Zhuqing Shi More articles by this author Andrew Rifkin More articles by this author S. Lilly Zheng More articles by this author Brian Helfand More articles by this author Alexander Glaser More articles by this author Jianfeng Xu More articles by this author Expand All Advertisement PDF downloadLoading ...

Genetics of Multiple System Atrophy and Progressive Supranuclear Palsy: A Systemized Review of the Literature

Multiple system atrophy (MSA) and progressive supranuclear palsy (PSP) are uncommon multifactorial atypical Parkinsonian syndromes, expressed by various clinical features. MSA and PSP are commonly considered sporadic neurodegenerative disorders; however, our understanding is improving of their genetic framework. The purpose of this study was to critically review the genetics of MSA and PSP and their involvement in the pathogenesis. A systemized literature search of PubMed and MEDLINE was performed up to 1 January 2023. Narrative synthesis of the results was undertaken. In total, 43 studies were analyzed. Although familial MSA cases have been reported, the hereditary nature could not be demonstrated. COQ2 mutations were involved in familial and sporadic MSA, without being reproduced in various clinical populations. In terms of the genetics of the cohort, synuclein alpha (SNCA) polymorphisms were correlated with an elevated likelihood of manifesting MSA in Caucasians, but a causal effect relationship could not be demonstrated. Fifteen MAPT mutations were linked with PSP. Leucine-rich repeat kinase 2 (LRRK2) is an infrequent monogenic mutation of PSP. Dynactin subunit 1 (DCTN1) mutations may imitate the PSP phenotype. GWAS have noted many risk loci of PSP (STX6 and EIF2AK3), suggesting pathogenetic mechanisms related to PSP. Despite the limited evidence, it seems that genetics influence the susceptibility to MSA and PSP. MAPT mutations result in the MSA and PSP pathologies. Further studies are crucial to elucidate the pathogeneses of MSA and PSP, which will support efforts to develop novel drug options.

Comparative cytogenetics of anembryonic pregnancies and missed abortions in human.

Miscarriage is an important problem in human reproduction, affecting 10-15 % of clinically recognized pregnancies. The cases of embryonic death can be divided into missed abortion (MA), for which the ultrasound sign of the embryo death is the absence of cardiac activity, and anembryonic pregnancy (AP) without an embryo in the gestational sac. The aim of this study was to compare the frequency of chromosomal abnormalities in extraembryonic tissues detected by conventional cytogenetic analysis of spontaneous abortions depending on the presence or absence of an embryo. This is a retrospective study of 1551 spontaneous abortions analyzed using GTG-banding from 1990 to 2022 (266 cases of AP and 1285 cases of MA). A comparative analysis of the frequency of chromosomal abnormalities and the distribution of karyotype frequencies depending on the presence of an embryo in the gestational sac was carried out. Statistical analysis was performed using a chi-square test with a p <0.05 significance level. The total frequency of chromosomal abnormalities in the study was 53.6 % (832/1551). The proportion of abnormal karyotypes in the AP and MA groups did not differ significantly and amounted to 57.1 % (152/266) and 52.9 % (680/1285) for AP and MA, respectively (p = 0.209). Sex chromosome aneuploidies and triploidies were significantly less common in the AP group than in the MA group (2.3 % (6/266) vs 6.8 % (88/1285), p = 0.005 and 4.9 % (13/266) vs 8.9 % (114/1285), p = 0.031, respectively). Tetraploidies were registered more frequently in AP compared to MA (12.4 % (33/266) vs. 8.2 % (106/1285), p = 0.031). The sex ratio among abortions with a normal karyotype was 0.54 and 0.74 for AP and MA, respectively. Thus, although the frequencies of some types of chromosomal pathology differ between AP and MA, the total frequency of chromosomal abnormalities in AP is not increased compared to MA, which indicates the need to search for the causes of AP at other levels of the genome organization, including microstructural chromosomal rearrangements, monogenic mutations, imprinting disorders, and epigenetic abnormalities.

Abnormal mTOR Signaling Pathway Activity in Autism Spectrum Disorders: Prospects of Mechanism-Based Therapy

Autism spectrum disorder (ASD) is a developmental disorder characterized by the early onset of communication, learning, and behavioral problems. The syndromic form of ASD is caused by monogenic mutations, in the case when it is not possible to find genetic or other known mechanisms, the term “idiopathic autism” is used. A significant part of both syndromic and idiopathic autism is associated with translational deregulation dependent on the mechanistic target of rapamycin (mTOR). In this review, we present both bioinformatic and experimental data that link the mTOR signaling pathway to maternal autoantibody-induced autism and childhood autoimmune neuropsychiatric disorders such as Sydenham’s chorea and pediatric autoimmune neuropsychiatric disorder associated with streptococcal infections (PANDAS). The need for ASD subtyping and the possibility of mechanism-based therapy with inhibitors of the mTOR signaling pathway are also discussed.

Abnormal mTOR Signaling Pathway Activity in Autism Spectrum Disorders: Prospects of Mechanism-Based Therapy

Autism spectrum disorder (ASD) is a developmental disorder characterized by the early onset of problems with communication, learning, and behavior. The syndromic form of ASD is caused by monogenic mutations. When it is not possible to find genetic or other known mechanisms, the term "idiopathic autism" is used. A significant part of both syndromic and idiopathic autism is associated with translational deregulation dependent on the mechanistic target of rapamycin (mTOR). In this review, we present both bioinformatic and experimental data that link the mTOR signaling pathway to maternal autoantibody related autism and childhood autoimmune neuropsychiatric disorders such as Sydenham's chorea and pediatric autoimmune neuropsychiatric disorder associated with streptococcal infections (PANDAS). The need for ASD subtyping and the prospects of mechanism-based therapy with inhibitors of the mTOR signaling pathway are also discussed.

Precision medicine for long QT syndrome: patient-specific iPSCs take the lead.

Long QT syndrome (LQTS) is a detrimental arrhythmia syndrome mainly caused by dysregulated expression or aberrant function of ion channels. The major clinical symptoms of ventricular arrhythmia, palpitations and syncope vary among LQTS subtypes. Susceptibility to malignant arrhythmia is a result of delayed repolarisation of the cardiomyocyte action potential (AP). There are 17 distinct subtypes of LQTS linked to 15 autosomal dominant genes with monogenic mutations. However, due to the presence of modifier genes, the identical mutation may result in completely different clinical manifestations in different carriers. In this review, we describe the roles of various ion channels in orchestrating APs and discuss molecular aetiologies of various types of LQTS. We highlight the usage of patient-specific induced pluripotent stem cell (iPSC) models in characterising fundamental mechanisms associated with LQTS. To mitigate the outcomes of LQTS, treatment strategies are initially focused on small molecules targeting ion channel activities. Next-generation treatments will reap the benefits from development of LQTS patient-specific iPSC platform, which is bolstered by the state-of-the-art technologies including whole-genome sequencing, CRISPR genome editing and machine learning. Deep phenotyping and high-throughput drug testing using LQTS patient-specific cardiomyocytes herald the upcoming precision medicine in LQTS.

Polygenic risk scores for complex diseases: Where are we now?

Polygenic risk scores for complex diseases: Where are we now?

Mitochondrial Genetic Background May Impact Statins Side Effects and Atherosclerosis Development in Familial Hypercholesterolemia

Heredity of familial hypercholesterolemia (FH) can present as a dominant monogenic disorder of polygenic origin or with no known genetic cause. In addition, the variability of the symptoms among individuals or within the same families evidence the potential contribution of additional factors than monogenic mutations that could modulate the development and severity of the disease. In addition, statins, the lipid-lowering drugs which constitute the first-line therapy for the disease, cause associated muscular symptoms in a certain number of individuals. Here, we analyze the evidence of the mitochondrial genetic variation with a special emphasis on the role of CoQ10 to explain this variability found in both disease symptoms and statins side effects. We propose to use mtDNA variants and copy numbers as markers for the cardiovascular disease development of FH patients and to predict potential statin secondary effects and explore new mechanisms to identify new markers of disease or implement personalized medicine strategies for FH therapy.

The Shank3Venus/Venus knock in mouse enables isoform-specific functional studies of Shank3a.

Shank3 is a scaffolding protein essential for the organization and function of the glutamatergic postsynapse. Monogenic mutations in SHANK3 gene are among the leading genetic causes of Autism Spectrum Disorders (ASD). The multiplicity of Shank3 isoforms seems to generate as much functional diversity and yet, there are no tools to study endogenous Shank3 proteins in an isoform-specific manner. In this study, we created a novel transgenic mouse line, the Shank3Venus/Venus knock in mouse, which allows to monitor the endogenous expression of the major Shank3 isoform in the brain, the full-length Shank3a isoform. We show that the endogenous Venus-Shank3a protein is localized in spines and is mainly expressed in the striatum, hippocampus and cortex of the developing and adult brain. We show that Shank3Venus/+ and Shank3Venus/Venus mice have no behavioral deficiency. We further crossed Shank3Venus/Venus mice with Shank3ΔC/ΔC mice, a model of ASD, to track the Venus-tagged wild-type copy of Shank3a in physiological (Shank3Venus/+) and pathological (Shank3Venus/ΔC) conditions. We report a developmental delay in brain expression of the Venus-Shank3a isoform in Shank3Venus/ΔC mice, compared to Shank3Venus/+ control mice. Altogether, our results show that the Shank3Venus/Venus mouse line is a powerful tool to study endogenous Shank3a expression, in physiological conditions and in ASD.

Genetic analysis of neurodevelopmental disorders in children

PurposeTo explore the genetic cause of children with unidentified etiology of neurodevelopmental disorders, thus providing references for the diagnosis, treatment and genetic counseling.Design and methodsChildren with neurodevelopmental disorders but unidentified etiology in the Child Healthcare Department, Children's Hospital of Nanjing Medical University from November 2018 to December 2021 were retrospectively analyzed. A total of 2 ml of peripheral venous blood was collected from the child and their parents for the whole exome sequencing (WES) and copy number variation (CNV) detection. Male children were subjected to fragile X syndrome testing to determine the genetic mutations. For those with positive results, Sanger sequencing was performed to explore the mutations in the gene sites and pedigrees.ResultsA total of 488 (33.5%) pathogenic variations were detected among 1,457 global developmental/intellectual disabilities (GDD/ID) children, including 362 (24.9%) cases of monogenic mutations, and 111 (7.6%) cases of chromosomal microdeletions or microduplications. There were 15/780 (1.92%) male children with fragile X syndrome. Single point mutations were detected in 277/362 (76.5%) and 85/362 (23.5%) male and female GDD/ID children, respectively, including 295 (81.5%) cases of missense mutations, 32 (8.8%) cases of frameshift mutations, 5 (2.2%) cases of non-sense mutations and 30 (8.3%) cases of splice site mutations. In addition, there were 166 (45.8%) cases of autosomal inheritance and 196 (54.2%) cases of X-linked inheritance. The X chromosome abnormalities were mostly observed in 362 GDD/ID children with monogenic mutations, including 15 cases of the AFF2 gene mutation, 13 cases of the MECP2 gene mutation and 12 cases of the HUWEI gene mutation. The CREBBP gene mutation was the most common autosome abnormality in GDD/ID children with monogenic mutations, which was detected in five cases. There were 74 cases of chromosomal microdeletions, 31 cases of chromosomal microduplications and six cases of both. A total of 114 novel pathogenic mutations responsible for GDD/ID were found, including four novel mutations in MECP2 and TRAPPC9 genes.ConclusionThe incidence of genetic abnormalities remains high in NDD children. Abundant novel mutations are responsible for GDD/ID in children, and can be used as references in the diagnosis of neurogenetic diseases.

Gene therapy in China: past, present, and future

Rapid advancements in gene therapy have greatly facilitated the treatments and cures for genetic disorders. In China, there are approximately 200 000 newborn babies each year with genetic disorders, 22% of which are caused by monogenic mutations. Birth defects, endocrine or metabolic diseases, nervous system diseases, and blood system diseases are the four most common genetic disorders. Additionally, gene therapies such as chimeric antigen receptor (CAR) T-cell immunotherapy have been increasingly reported as effective treatments for diseases such as cancer and HIV infection.

In vitro systems to study inborn errors of immunity using human induced pluripotent stem cells.

In the last two decades, the exponential progress in the field of genetics could reveal the genetic impact on the onset and progression of several diseases affecting the immune system. This knowledge has led to the discovery of more than 400 monogenic germline mutations, also known as "inborn errors of immunity (IEI)". Given the rarity of various IEI and the clinical diversity as well as the limited available patients' material, the continuous development of novel cell-based in vitro models to elucidate the cellular and molecular mechanisms involved in the pathogenesis of these diseases is imperative. Focusing on stem cell technologies, this review aims to provide an overview of the current available in vitro models used to study IEI and which could lay the foundation for new therapeutic approaches. We elaborate in particular on the use of induced pluripotent stem cell-based systems and their broad application in studying IEI by establishing also novel infection culture models. The review will critically discuss the current limitations or gaps in the field of stem cell technology as well as the future perspectives from the use of these cell culture systems.

Expanded Autoreactive Transitional B Cells in Monogenic Common Variable Immunodeficiency

Introduction Autoimmune cytopenias (AIC) are prevalent in patients with primary immune deficiencies, including common variable immunodeficiency (CVID). They are often refractory and difficult to treat, and patients with autoimmune complications of CVID have a higher risk of mortality than those with an infectious phenotype (Farmer et al. Front Immnol 2017; Resnick et al. Blood 2021). The pathogenesis of AIC in CVID is not well understood, making diagnosis and prognostication challenging. We previously observed a block in development from transitional to follicular B cells in patients with activated PI3K delta syndrome (APDS), a monogenic form of CVID (Farmer et al. Sci Signal 2019). We have now investigated a larger cohort with various causes of monogenic CVID (45% with AIC). We sought to determine if a block in B cell development extended beyond APDS to other patients with monogenic CVID and autoimmunity. We aimed to identify a link between the transitional B cell block and a break in tolerance, as transitional B cells are immature cells with self-reactive properties prior to differentiating into mature follicular B cells (Allman et al. Curr Opin Immunol 2008). Thus, we performed flow cytometry using the 9G4 monoclonal antibody to identify B cells containing the 9G4 idiotope of the immunoglobulin heavy chain VH4-34 gene. This idiotope is associated with autoimmunity (Cappione et al. J Clin Invest 2005; Pugh-Bernard et al. J Clin Invest 2001). Utilizing single-cell Cellular Indexing of Transcriptomes and Epitopes by Sequencing (scCITE-Seq) and B cell receptor (BCR) repertoire, we analyzed patients with APDS to further define heterogeneous B cell populations. We leveraged our access to biospecimens and clinical data from patients with monogenic CVID to study pathologic B cells. Methods Patients were recruited in accordance with the Partners Institutional Review Board. The cohort was queried for cytopenias, including immune thrombocytopenia (ITP), autoimmune hemolytic anemia (AIHA), and autoimmune neutropenia (AIN), identified by positive history on physician chart review or otherwise captured by review of all clinical details in the Electronic Medical Record. We performed flow cytometry on peripheral blood mononuclear cells with a detailed B cell panel including the 9G4 antibody. We included 20 patients with monogenic CVID (PI3K (n=4), NFKB1 (n=5), and CTLA4 mutations (n=11)) and 12 healthy controls. Nine patients had AIC (ITP (n=3), ITP + AIHA (n=4), ITP + AIN (n=1), ITP + AIHA + AIN (n=1)). We sequenced total CD19+ B cells from 4 patients with APDS and 4 controls using scCITE-Seq. With the Immcantation software suite, we identified clonal clusters based on BCR sequences and constructed maximum parsimony lineage trees for clusters with >5 unique sequences. Results Patients with monogenic CVID and AIC had marked expansion of 9G4+ B cells compared to healthy controls. There was a statistically significant (p<0.05) increase in the percent of 9G4+ B cells in patients with AIC compared to controls in the transitional T1/2, T3a, and T3b B cell subsets, as well as double negative, follicular, activated naïve, marginal zone precursor, and marginal zone subsets. This expansion was particularly pronounced in the transitional B cell population (p<0.001). ScCITE-Seq identified 19 B cell clusters. Clonal B cell populations separated into three predominant IgM isotype clusters in healthy controls (5, 9, and 15), while in patients with APDS there were additional clusters identified, including IgM isotype clusters 0, 2, 3, 10, 13, 17 and IgG isotype clusters 4, 6, 10, 12, and 16 (Figure 1). This suggests that there are novel, isotype-switched B cell states present in patients with APDS that are not present in healthy controls. Conclusions In this study, we investigated B cell populations in patients with monogenic CVID and autoimmunity. We found expansion of transitional B cells characterized by 9G4 positivity. These transitional cells may fail to be eliminated because they are aberrantly activated and differentiate into self-reactive activated B cells and plasmablasts that secrete pathogenic autoantibodies. ScCITE-Seq revealed novel B cell subsets in patients with APDS and could represent an altered state of B cell biology that leads to pathologic autoantibody production and clinical autoimmunity. These results improve identification of heterogeneous B cell subsets and connectivity between subsets. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cohesin Dependency in Npm1c/+ and Flt3-ITD Mutant Hematopoietic Stem and Progenitor Cells

Acute myeloid leukemia (AML) is an aggressive myeloid malignancy with high relapse (40-60%) and low median survival rate (5-7 months post treatment in relapse setting), particularly for patients over 65 years old. Genomic investigations of AML have identified recurrent mutations, such as FMS-like tyrosine kinase 3 (FLT3, 20-25%) and Nucleophosmin (NPM1, 30%). When mutated, FLT3 acquires an internal tandem duplication (FLT3-ITD) and causes aberrant STAT5/AKT signaling. For NPM1, mutation frequently occurs at nuclear export signal, which results in cytoplasmic localization (NPM1c). In AML, these driver mutations co-occur with other genetic lesions, particularly in epigenetic regulators. Clincally, patients with FLT3-ITD mutations have a very poor prognosis. While the pathophysiology is not completely understood, direct inhibition of FLT3 by tyrosine kinase inhibitors have, to-date, been underwhelming. In contrast, the NPM1c mutation in AML patients is favorable and chemotherapy sensitive when NPM1c is the sole driver mutation. Yet, co-mutation of NPM1c with other known AML mutations, such as Flt3-ITD, negates this favorable prognosis. STAG2 (Stromal Antigen 2) is a member of cohesin complex that is recurrently mutated in >10 cancers and is essential in maintaining the integrity of the 3-dimensional genome partitioning structure known as topologically structural domains (TADs). Previously, our work has demonstrated that depletion of various cohesin factors, including Stag2, leads to increased hematopoietic stem and progenitor cell (HSPC) self-renewal and myeloid-biased differentiation. Loss of Stag2 leads to impairment of sub-TADs and affects the capability of key hematopoietic transcription factors, such as PU.1, to access and engage their target genes. In the context of AML, disruption of TADs can lead to altered transcriptional output and cellular behavior. Mutation of STAG2 frequently arises in AML (16%) and co-occurs with NPM1 and FLT3 oncogenes. To examine the consequences of co-occurring Stag2/Npm1c or Stag2/Flt3-ITD mutations, we generated two compound mutant murine models crossed to conditional alleles of Stag2, Npm1c and constitutively active Flt3-ITD in the Ubc-CreER background. At 6-8 weeks of age, tamoxifen was administered to mice harboring Stag2/Npm1c or Stag2/Flt3-ITD mutations to delete Stag2 or activate Npm1c alleles respectively, while the Flt3-ITD mutation is active since birth. Immunophenotyping analysis was performed at 4 weeks post mutation activation in the bone marrow and peripheral blood. For mice carrying double Stag2/Npm1c mutation, there was no change in the number of stem cell population (HSCLT). In contrast, comparing to Npm1c/+ mice, Stag2-/-Npm1c/+ mice had an elevated myeloid-biased multipotent progenitor population (MPP3, LSK flk2- CD150- CD48+). However, there was no change in granulocyte-monocyte progenitor (GMP) number (Figure 1A). To determine if the myeloid bias was due to lack of the entire cohesin complex, we generated the Smc3/Npm1c mutant mice, where both Stag1-cohesin and Stag2-cohesin complexes were partially depleted upon heterozygous deletion of Smc3. Interestingly, there were no changes in MPP3 population in Smc3+/-Npm1c/+ mice, pointing to a Stag2-cohesin specific phenomenon. In the Stag2/Flt3-ITD co-mutation model, loss of Stag2-cohesin resulted in no changes in MPP3 cells compared to monogenic Flt3-ITD mutation. However, we did observe a normalization of GMPs, suggesting that Stag2-cohesin loss is crucial for abnormal myeloid differentiation in Flt3-ITD background (Figure 1B). In order to evaluate whether the order of mutations impacted immunophenotypic aberrancy, we additionally generated a stepwise Stag2/Flt3-ITD mutational model by deleting Stag2 first (via Mx1-Cre) for 4 weeks followed by tamoxifen-inducible Frt activated Flt3-ITD mutation (Flt3 frt-ITD/+) for 4 weeks. Interestingly, there was no difference in the normalization effect between constitutive and conditional activation of Flt3-ITD mutation, suggesting the loss of Stag2-cohesin is not required prior to Flt3-ITD acquisition. Further characterization of these co-mutational murine models is in progress. With these models, we aim to uncover the dependency of Stag2-cohesin in leukemia maintenance. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CRISPR/nCas9-Based Genome Editing on GM2 Gangliosidoses Fibroblasts via Non-Viral Vectors.

The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the β-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the β-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay–Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the β-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the β-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.

A novel multifunctional haplotyping-based preimplantation genetic testing for different genetic conditions.

Is there an efficient and cost-effective detection platform for different genetic conditions about embryos? A multifunctional haplotyping-based preimplantation genetic testing platform was provided for detecting different genetic conditions. Genetic disease and chromosomal rearrangement have been known to significantly impact fertility and development. Therefore, preimplantation genetic testing for aneuploidy (PGT-A), monogenic disorders (PGT-M) and structural rearrangements (PGT-SR), a part of ART, has been presented together to minimize the fetal genetic risk and increase pregnancy rate. For patients or their families who are suffering from chromosome abnormality, monogenic disease, unexplained repeated spontaneous abortion or implantation failure, after accepting genetic counseling, they may be suggested to accept detection from more than one PGT platforms about the embryos to avoid some genetic diseases. However, PGT platforms work through different workflows. The high costliness, lack of material and long-time operation of combined PGT platforms limit their application. All 188 embryonic samples from 43 families were tested with HaploPGT platform, and most of their genetic abnormalities had been determined by different conventional PGT methods beforehand. Among them, there were 12 families only carrying structural rearrangements (115 embryos) in which 9 families accepted implantation and 5 families had normal labor ART outcomes, 7 families only carrying monogenic diseases (26 embryos) and 3 families carrying both structural rearrangements and monogenic diseases (26 embryos). Twelve monopronucleated zygotes (1PN) samples and 9 suspected triploid samples were collected from 21 families. Here, we raised a comprehensive PGT method called HaploPGT, combining reduced representation genome sequencing, read-count analysis, B allele frequency and haplotyping analysis, to simultaneously detect different genetic disorders in one single test. With 80 million reads (80M) genomic data, the proportion of windows (1 million base pairs (Mb)) containing two or more informative single nucleotide polymorphism (SNP) sites was 97.81%, meanwhile the genotyping error rate stabilized at a low level (2.19%). Furthermore, the informative SNPs were equally distributed across the genome, and whole-genomic haplotyping was established. Therefore, 80M was chosen to balance the cost and accuracy in HaploPGT. HaploPGT was able to identify abnormal embryos with triploid, global and partial loss of heterozygosity, and even to distinguish parental origin of copy number variation in mosaic and non-mosaic embryos. Besides, by retrospectively analyzing 188 embryonic samples from 43 families, HaploPGT revealed 100% concordance with the available results obtained from reference methods, including PGT-A, PGT-M, PGT-SR and PGT-HLA. Despite the numerous benefits HaploPGT could bring, it still required additional family members to deduce the parental haplotype for identifying balanced translocation and monogenic mutation in tested embryos. In terms of PGT-SR, the additional family member could be a reference embryo with unbalanced translocation. For PGT-M, a proband was normally required. In both cases, genomic information from grandparents or parental siblings might help for haplotyping theoretically. Another restriction was that haploid, and diploid resulting from the duplication of a haploid, could not be told apart by HaploPGT, but it was able to recognize partial loss of heterozygosity in the embryonic genome. In addition, it should be noted that the location of rearrangement breakpoints and the situation of mutation sites were complicated, which meant that partial genetic disorders might not be completely detected. HaploPGT is an efficient and cost-effective detection platform with high clinical value for detecting genetic status. This platform could promote the application of PGT in ART, to increase pregnancy rate and decrease the birth of children with genetic diseases. This study was supported by grants from the National Natural Science Foundation of China (81873478, to L.H.), National Key R&D Program of China (2018YFC1003100, to L.H.), the Natural Science Foundation of Hunan Province (Grant 2022JJ30414, to P.X.), Hunan Provincial Grant for Innovative Province Construction (2019SK4012) and the Scientific Research Foundation of Reproductive and Genetic Hospital of China International Trust & Investment Corporation (CITIC)-Xiangya (YNXM-201910). Haplotyping analysis has been licensed to Basecare Co., Ltd. L.K., Y.M., K.K., D.Z., N.L., J.Z. and R.D. are Basecare Co., Ltd employees. The other authors declare no competing interests. N/A.

Intracellular Sensing of DNA in Autoinflammation and Autoimmunity.

Evidence has shown that DNA is a pathogen-associated molecular pattern, posing a unique challenge in the discrimination between endogenous and foreign DNA. This challenge is highlighted by certain autoinflammatory diseases that arise from monogenic mutations and result in periodic flares of inflammation, typically in the absence of autoantibodies or antigen-specific T lymphocytes. Several autoinflammatory diseases arise due to mutations in genes that normally prevent the accrual of endogenous DNA or are due to mutations that cause activation of intracellular DNA-sensing pathway components. Evidence from genetically modified murine models further support an ability of endogenous DNA and DNA sensing to drive disease pathogenesis, prompting the question of whether endogenous DNA can also induce inflammation in human autoimmune diseases. In this review, we discuss the current understanding of intracellular DNA sensing and downstream signaling pathways as they pertain to autoinflammatory disease, including the development of monogenic disorders such as Stimulator of interferon genes-associated vasculopathy with onset in infancy and Aicardi-Goutières syndrome. In addition, we discuss systemic rheumatic diseases, including certain forms of systemic lupus erythematosus, familial chilblain lupus, and other diseases with established links to intracellular DNA-sensing pathways, and highlight the lessons learned from these examples as they apply to the development of therapies targeting these pathways.